Laboratory of pharmacokinetics, Department of Biological Testing, Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences in Puschino, Pushchino, Moscow region 142290, Russia.
Department of Cell Signaling, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia.
Biomolecules. 2020 Jul 10;10(7):1025. doi: 10.3390/biom10071025.
N-terminal myristoylation is a common co-and post-translational modification of numerous eukaryotic and viral proteins, which affects their interaction with lipids and partner proteins, thereby modulating various cellular processes. Among those are neuronal calcium sensor (NCS) proteins, mediating transduction of calcium signals in a wide range of regulatory cascades, including reception, neurotransmission, neuronal growth and survival. The details of NCSs functioning are of special interest due to their involvement in the progression of ophthalmological and neurodegenerative diseases and their role in cancer. The well-established procedures for preparation of native-like myristoylated forms of recombinant NCSs via their bacterial co-expression with N-myristoyl transferase from Saccharomyces cerevisiae often yield a mixture of the myristoylated and non-myristoylated forms. Here, we report a novel approach to preparation of several NCSs, including recoverin, GCAP1, GCAP2, neurocalcin δ and NCS-1, ensuring their nearly complete N-myristoylation. The optimized bacterial expression and myristoylation of the NCSs is followed by a set of procedures for separation of their myristoylated and non-myristoylated forms using a combination of hydrophobic interaction chromatography steps. We demonstrate that the refolded and further purified myristoylated NCS-1 maintains its Са-binding ability and stability of tertiary structure. The developed approach is generally suited for preparation of other myristoylated proteins.
N-端豆蔻酰化是许多真核生物和病毒蛋白常见的共翻译和翻译后修饰，它影响其与脂质和伴侣蛋白的相互作用，从而调节各种细胞过程。其中包括神经元钙传感器（NCS）蛋白，介导钙信号在广泛的调节级联中的转导，包括接收、神经传递、神经元生长和存活。由于 NCSs 参与眼科和神经退行性疾病的进展及其在癌症中的作用，因此它们的功能细节特别令人感兴趣。通过与酿酒酵母 N-豆蔻酰转移酶在细菌中共表达来制备天然样豆蔻酰化形式的重组 NCSs 的既定程序通常会产生豆蔻酰化和非豆蔻酰化形式的混合物。在这里，我们报告了一种制备几种 NCSs（包括 recoverin、GCAP1、GCAP2、神经钙蛋白 δ 和 NCS-1）的新方法，确保它们几乎完全豆蔻酰化。优化细菌表达和 NCSs 的豆蔻酰化后，采用一系列疏水性相互作用色谱步骤的组合来分离其豆蔻酰化和非豆蔻酰化形式。我们证明了重折叠和进一步纯化的豆蔻酰化 NCS-1 保持其 Са 结合能力和三级结构稳定性。所开发的方法通常适用于制备其他豆蔻酰化蛋白。
